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目的探讨在灌注型生物反应器中,大段磷酸三钙(β-TCP)载体内不同流速对人骨髓间充质干细胞(humanmesenchymal stem cells,hMSCs)增殖及成骨分化的影响。方法利用灌注型生物反应器对复合了hMSCs的大段β-TCP载体分别以3、6、9 mL/min的流速培养15 d,通过葡萄糖消耗量、细胞活力(MTT比色法)以及扫描电镜(SEM)观测细胞在载体内的增殖情况;以Real-time PCR检测成骨分化相关基因的表达。结果各组葡萄糖的日消耗量随培养时间的延长而增加,初期9 mL/min组细胞增殖明显快于3、6 mL/min组(P<0.001),但是灌注培养后期6 mL/min组的细胞增殖快于9、3 mL/min组(P<0.05)。灌注培养15 d后,6 mL/min组细胞活力显著高于3、9 mL/min组(P<0.001)。SEM观察发现3组β-TCP载体内均形成复合细胞层,3 mL/min组细胞层呈疏松的簇状,6 mL/min组复合细胞层部分呈膜片状,9 mL/min组复合细胞层多数呈膜片状。在进行成骨诱导灌注培养15 d后,6、9 mL/min组碱性磷酸酶(ALP)及骨桥蛋白(OP)的表达均显著高于3 mL/min组(P<0.01);3、6 mL/min组骨钙素(OC)的表达基本相同(P>0.05),而9 mL/min组OC的表达量则显著高于另外两组(P<0.001)。结论在利用灌注型生物反应器对hMSCs进行灌注培养的早期,9 mL/min的流速最有利于hMSCs的增殖,而晚期6 mL/min的流速最有利于hMSCs的增殖。β-TCP载体内流体剪切应力(flow shear stress,FSS)随灌注流速的增加而增加,适当的FSS可促进细胞外基质的合成和分布,并增加成骨相关基因的表达。
Objective To investigate the effects of different flow rates on the proliferation and osteogenic differentiation of human mesenchymal stem cells (hMSCs) in a perfusion bioreactor. Methods The perfusion-type bioreactor was used to culture a large section of β-TCP vector with hMSCs at 15, 6 and 9 mL / min respectively for 15 days. The glucose consumption, cell viability (MTT colorimetry) and scanning electron microscopy (SEM) to observe the proliferation of cells in the vector; Real-time PCR detection of osteogenic differentiation-related gene expression. Results The daily consumption of glucose in each group increased with the prolongation of culture time. The initial cell proliferation rate in 9 mL / min group was significantly faster than that in 3,6 mL / min group (P <0.001) Cell proliferation was faster than 9,3 mL / min group (P <0.05). After 15 days of perfusion culture, the viability of 6 mL / min group was significantly higher than that of 3, 9 mL / min group (P <0.001). The results of SEM showed that the composite cell layer was formed in all 3 groups of β-TCP vector, the cell layer in 3 mL / min group was loose clusters, the membrane layer in 6 mL / min group was patchy, The majority of layers were patchy. After osteogenic induction perfusion culture for 15 days, ALP and OP expression in 6 and 9 mL / min group were significantly higher than those in 3 mL / min group (P <0.01); 3 , While the expression of osteocalcin (OC) in 6 mL / min group was basically the same (P> 0.05). The expression of OC in 9 mL / min group was significantly higher than that in the other two groups (P <0.001). Conclusion In the early perfusion culture of hMSCs by perfusion bioreactor, the flow rate of 9 mL / min is the best for the proliferation of hMSCs, while the flow rate of 6 mL / min in late stage is the most conducive to hMSCs proliferation. The flow shear stress (FSS) of β-TCP carrier increased with the increase of perfusion flow rate. Appropriate FSS could promote the synthesis and distribution of extracellular matrix and increase the expression of osteogenesis-related genes.